Published May 1, 2000
The Role of the Putative Inactivation Lid in Sodium Channel Gating
Current Immobilization
Michael F. Sheets,* John W. Kyle,‡ and Dorothy A. Hanck§
From the *Nora Eccles Harrison Cardiovascular Research & Training Institute and The Department of Internal Medicine, University of
Utah, Salt Lake City, Utah 84112; and ‡Department of Neurobiology, Pharmacology, and Physiology, and §Department of Medicine,
University of Chicago, Chicago, Illinois 60637
key words:
sodium channel • gating charge • inactivation • site-3 toxin • immobilization
I N T R O D U C T I O N
The primary process by which voltage-gated sodium
channels become nonconductive after opening in response to a strong step depolarization is called inactivation. Armstrong and Bezanilla (1977) were the first to
demonstrate the relationship between fast inactivation
of sodium ionic current (INa) and measurements of sodium channel gating currents (Ig), small asymmetrical
currents that result from the movement of charged portions of the channel in response to changes in the electrical field. They showed that the time course of development of a slow component in Ig recorded during repolarization at very negative potentials correlated with the
time course of INa inactivation. When the membrane potential was repolarized to less negative potentials, the
slow component became too small and too slow to be accurately measured, and they described the gating charge
as becoming “immobilized.” Nearly 60% of the total sodium channel gating charge could become immobilized
in squid giant axon, and similar values have been reported in other preparations (Meves and Vogel, 1977;
Nonner, 1980; Starkus et al., 1981; Greeff et al., 1982).
Since the first cloning of a voltage-gated sodium channel by Noda et al. (1984), specific portions of the channel’s sequence have become associated with specialized
channel functions. The voltage sensors have been shown
to reside, in large part, in the fourth transmembrane
spanning segment (S4) in each of four domains that form
the ␣ subunit. Part of the fast inactivation process was localized to the intracellular region formed by the linker
between domains III and IV by Vassilev et al. (1988), who
used antibodies directed to this region and by Stühmer et
al. (1989), who noted a prolongation of INa decay when
the linker was cut and channels expressed in two pieces.
This intracellular linker has been called the “inactivation
lid” after mutation of the three adjacent amino acids, isoleucine, phenylalanine, and methionine (IFM),1 to QQQ
1Abbreviations
Portions of this work were previously published in abstract form
(Sheets, M.F., J.W. Kyle, and D.A. Hanck. 2000. Biophys. J. 76:A193).
Address correspondence to Michael Sheets, M.D., CVRTI, Bldg.
500, 95 South 2000 East, University of Utah, Salt Lake City, UT
84112. Fax: 801-581-3128; E-mail: [email protected]
609
used in this paper: Ap-A, Anthopleurin-A; hH1a, human
heart sodium channel; ICM-hH1a, human heart sodium channel
with F1485C mutation; IFM, isoleucine, phenylalanine, and methionine; MTSET, 2-(trimethylammonium)ethyl methanethiosulfonate;
OFF charge, gating charge upon repolarization; ON charge, gating
charge upon depolarization.
J. Gen. Physiol. © The Rockefeller University Press • 0022-1295/2000/05/609/11 $5.00
Volume 115 May 2000 609–619
http://www.jgp.org/cgi/content/full/115/5/609
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a b s t r a c t We investigated the contribution of the putative inactivation lid in voltage-gated sodium channels to gating
charge immobilization (i.e., the slow return of gating charge during repolarization) by studying a lid-modified mutant of
the human heart sodium channel (hH1a) that had the phenylalanine at position 1485 in the isoleucine, phenylalanine,
and methionine (IFM) region of the domain III–IV linker mutated to a cysteine (ICM-hH1a). Residual fast inactivation of
ICM-hH1a in fused tsA201 cells was abolished by intracellular perfusion with 2.5 mM 2-(trimethylammonium)ethyl methanethiosulfonate (MTSET). The time constants of gating current relaxations in response to step depolarizations and gating charge–voltage relationships were not different between wild-type hH1a and ICM-hH1aMTSET. The time constant of
the development of charge immobilization assayed at ⫺180 mV after depolarization to 0 mV was similar to the time constant of inactivation of INa at 0 mV for hH1a. By 44 ms, 53% of the gating charge during repolarization returned slowly;
i.e., became immobilized. In ICM-hH1aMTSET, immobilization occurred with a similar time course, although only 31% of
gating charge upon repolarization (OFF charge) immobilized. After modification of hH1a and ICM-hH1aMTSET with Anthopleurin-A toxin, a site-3 peptide toxin that inhibits movement of the domain IV-S4, charge immobilization did not occur for conditioning durations up to 44 ms. OFF charge for both hH1a and ICM-hH1aMTSET modified with Anthopleurin-A toxin were similar in time course and in magnitude to the fast component of OFF charge in ICM-hH1aMTSET in
control. We conclude that movement of domain IV-S4 is the rate-limiting step during repolarization, and it contributes to
charge immobilization regardless of whether the inactivation lid is bound. Taken together with previous reports, these
data also suggest that S4 in domain III contributes to charge immobilization only after binding of the inactivation lid.
Published May 1, 2000
M E T H O D S
cDNA Clones
In hH1a (kindly provided by H. Hartmann and A. Brown (see
Hartmann et al., 1994) the phenylalanine in the IFM motif at
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Gating Current Immobilization
amino acid position 1485 (Fig. 1) was mutated to a cysteine by
four-primer PCR (Higuchi et al., 1988; Ho et al., 1989). The insert containing the mutated site was confirmed by sequencing.
Because anecdotal evidence suggested that cell survival of inactivation-impaired mutants could be increased by blocking sodium
channels during culturing, we increased the sensitivity of the
ICM-hH1a channel to block by tetrodotoxin by mutating the cysteine at position 373 to a tyrosine (C373Y) (Satin et al., 1994;
Chen et al., 1996). For expression of both hH1a and ICM-hH1a
in mammalian cells, the cDNAs were subcloned directionally into
the mammalian expression vector pRcCMV (Invitrogen Corp.).
Cell Preparation
Multiple tsA201 cells (SV40-transformed HEK293 cells) or multiple HEK293 cells were fused together using polyethylene glycol,
as previously described (Sheets et al., 1996). After fusion, the
cells were placed in cell culture for several days to allow for membrane remodeling, and then they were transiently transfected using a calcium phosphate precipitation method (GIBCO BRL).
For some experiments with wild-type hH1a, HEK293 cells that
stably expressed hH1a were fused before transient transfection
with cDNA coding for hH1a. ICM-hH1a expression appeared to
be increased by the addition of 300 nM tetrodotoxin to the culture media 1 d after transfection. 3–6 d after transfection, fused
cells were detached from culture dishes with trypsin-EDTA solution (GIBCO BRL) and studied electrophysiologically.
Recording Technique, Solutions, and Experimental Protocols
Recordings were made using a large bore, double-barreled glass
suction pipette for both voltage clamp and internal perfusion, as
previously described (Hanck et al., 1990; Sheets et al., 1996). I Na
was measured with a virtual ground amplifier (OPA-101; BurrBrown) using a 2.5 M⍀ feedback resistor. Voltage protocols were
imposed from a 16-bit DA converter (Masscomp 5450; Concurrent Computer) over a 30/1 voltage divider. Data were filtered by
the inherent response of the voltage-clamp circuit (corner frequency near 125 kHz) and recorded with a 16-bit AD converter
on a Masscomp 5450 at 200 kHz. A fraction of the current was
fed back to compensate for series resistance. Temperature was
controlled using a Sensortek thermoelectric stage (TS-4; Physiotemp Instruments, Inc.) mounted beneath the bath chambers
and it typically varied ⬍0.5⬚C during an experimental set. Cells
were usually studied at 12⬚–13⬚C.
A cell was placed in the aperture of the pipette and, after a
high resistance seal formed, the cell membrane inside the pipette was disrupted with a manipulator-controlled platinum wire.
Voltage control was assessed by evaluating the time course of the
capacitive current and the steepness of the negative slope region
of the peak current–voltage relationship as per criteria previously
established (Hanck and Sheets, 1992). To allow for full sodium
channel availability, the holding membrane potential was set to
⫺150 mV. Ig protocols typically contained four repetitions at
each test voltage that were one fourth of a 60 Hz cycle out of
phase to maximize rejection of this frequency and to improve the
signal-to-noise ratio.
The control extracellular solution for INa measurements contained (mM): 15 Na⫹, 185 TMA⫹, 2 Ca2⫹, 200 MES⫺, and 10
HEPES, pH 7.2; and the intracellular solution contained: 200
TMA⫹, 200 F⫺, 10 EGTA, and 10 HEPES, pH 7.2. For measurement of Ig, the intracellular solution contained (mM): 200 TMA ⫹,
50 mM F⫺, 150 mM MES⫺, 1 mM BAPTA (1,2-bis 2-aminophenoxy-ethane-N,N,N ⬘,N-tetraacetic acid; Sigma Chemical Co.), 10
HEPES, and 10 EGTA, pH 7.2, 10 ␮M saxitoxin (Calbiochem
Corp.), and the extracellular solution had Na ⫹ replaced with
TMA⫹. Reduction of intracellular F⫺ and inclusion of BAPTA re-
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removed fast inactivation (see Fig. 1) in rat brain IIa
(West et al., 1992) and in human heart (hH1a) (Hartmann et al., 1994). Additional studies have shown that
the voltage sensor formed by domain IV–S4 has a unique
role in channel inactivation (Chahine et al., 1994; Yang
and Horn, 1995; Chen et al., 1996; Yang et al., 1996; Kontis et al., 1997; Kuhn and Greeff, 1999). In our previous
studies, using a site-3 peptide toxin that binds to the outside of sodium channels (Rogers et al., 1996; Benzinger et
al., 1998), we identified a component of sodium channel
Ig that is associated with inactivation from the open state
(Hanck and Sheets, 1995; Sheets and Hanck, 1995), and
we showed that the gating current component resulted
from movement of the S4 in domain IV (Sheets et al.,
1999). Recently, Cha et al. (1999), using site-directed fluorescent labeling of human skeletal muscle sodium channels, demonstrated that the S4 segments in domains III
and IV, but not those in domains I and II, contributed to
charge immobilization.
To investigate the role of the inactivation lid in charge
immobilization, we studied the human cardiac sodium
channel that had the phenylalanine at amino acid position 1485 in the IFM motif mutated to a cysteine (ICMhH1a). The mutant sodium channel was transiently expressed in fused mammalian cells (Sheets et al., 1996),
and the cysteine was modified by intracellular application
of 2-(trimethylammonium)ethyl methanethiosulfonate
(MTSET) to remove residual fast inactivation (ICMhH1aMTSET) (Kellenberger et al., 1996; Chahine et al.,
1997; Vedantham and Cannon, 1998). Our results showed
that the gating charge-voltage (Q-V) relationship and the
dominant time constants of Ig relaxations recorded during
step depolarizations were unaltered regardless of whether
the inactivation lid could bind to its receptor. In the absence of a functioning inactivation lid, charge immobilization still occurred in ICM-hH1aMTSET with a time course
similar to that in wild-type hH1a, but only 31% of gating
charge upon repolarization (OFF charge) became immobilized, compared with 53% in hH1a. Experiments with
Anthopleurin-A (Ap-A) toxin, a site-3 toxin that inhibits
movement of the S4 in domain IV, abolished charge immobilization for both hH1a and ICM-hH1aMTSET. These
data indicate that movement of the S4 in domain IV is the
rate-limiting step in repolarization, and contributes to
charge immobilization regardless of whether the inactivation lid is bound. Taken together with previous reports
(Cha et al., 1999), these data also suggest that the S4 in domain III contributes to charge immobilization only after
binding of the inactivation lid.
Published May 1, 2000
Data were analyzed and graphed on a SUN Sparcstation using
SAS (Statistical Analysis System). Unless otherwise specified,
summary statistics are expressed as means ⫾ 1 SD. Regression parameters are reported as the estimate and the standard error of
the estimate (S.E.E.), and figures show means ⫾ SEM.
R E S U L T S
Intracellular MTSET Modification of ICM-hH1a
Figure 1. Predicted membrane topology of the sodium channel
showing the four domains, each with six transmembrane segments,
the charged residues in the fourth membrane–spanning regions
(S4), and the intracellular locations of both the NH 2 and COOH
termini. The phenylalanine at position 1485 in the IFM region of
the linker between domains III and IV was mutated to a cysteine.
duced the magnitude of endogenous ionic currents that occasionally interfered with recordings. Anthopleurin A toxin (Sigma
Chemical Co.) was used at a concentration of 1 ␮M, which is
three orders of magnitude greater than the Kd (Hanck and
Sheets, 1995; Khera et al., 1995). For experiments in which cells
were perfused internally with MTSET (Toronto Research Chemicals), it was dissolved in intracellular solutions ⵑ1 min before use.
Leak resistance was calculated as the reciprocal of the linear conductance between ⫺190 and ⫺110 mV, and cell capacitance was
measured from the integral of the current responses to voltage
steps between ⫺150 and ⫺190 mV. Peak INa was taken as the
mean of four data samples clustered around the maximal value
of data digitally filtered at 5 kHz and leak corrected by the
amount of the calculated time-independent linear leak. Data
were capacity corrected using 4–16 scaled current responses recorded from voltage steps between ⫺150 and ⫺190 mV. All Ig
were leak corrected by the mean of 2–4 ms of data, beginning at
least 8 ms after the change in potential. For ON-Ig, this was typically at 8 ms, and 10 ms for OFF-Ig.
To determine time constants of Ig decay, current traces were
trimmed until the decay phase was clearly apparent, and then fit
by a sum of exponentials with DISCRETE (Provencher, 1976), a
program that provides a modified F statistic to evaluate the number of exponential components that best describe the data. To
calculate the fraction of gating charge associated with each of the
exponential components, it was necessary take into account the
fact that the voltage clamp was not instantaneous. We first calculated the total OFF charge during each repolarization step, and
then extrapolated the exponential curve backwards from the first
point used for fitting until the total charge from the fitted curve
equaled the total OFF charge for that voltage step. This method
was a compromise between extrapolation of the data back to the
start of the repolarization step, which would have resulted in over
estimation of the contribution of the fast-time constant, and no
extrapolation at all, which would have over estimated the contribution of the slow-time constant.
Charge-voltage relationships were fit with a simple Boltzmann
distribution as follows:
Q max
-,
Q = -------------------------V –V
t
1+e
(1)
1⁄2
-----------------------s
where Q is the charge during depolarizing step, Qmax is the maximum charge, Vt is the test potential, V1/2 is the half point of the
relationship, and s is the slope factor in millivolts. For comparison
between cells, fractional Q was calculated as Q/Qmax for each cell.
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Sheets et al.
ON-Gating Current Studies
If the putative inactivation lid were outside the voltage
field and all voltage sensors had completed their translocation before the binding of the inactivation lid to its
receptor, then Ig measured during step depolarizations
should be insensitive to whether the inactivation lid
can become bound to its receptor. Fig. 3 shows capacity
and leak-corrected Ig traces and their corresponding integrals for typical cells expressing hH1a and ICMhH1aMTSET. Ig decays were fit by a sum of up to two exponentials, and a two-time-constant fit was accepted
when it produced a statistically significant F statistic
(Provencher, 1976). The dominant time constant was
assigned as the one making the larger contribution to
total gating charge. For hH1a, two exponentials fit better 53% of the time, while for ICM-hH1aMTSET two exponentials fit better 77% of the time. When there was a
second exponential, it contributed only 9% ⫾ 8% (n ⫽
5) to the gating charge in hH1a and 15% ⫾ 11% (n ⫽
4) to the charge in ICM-hH1aMTSET channels. There
was no difference between the dominant time constants for the two channels at any of the test potentials
(Fig. 3 C). In addition, the Q-V relationships were
nearly identical between hH1a and ICM-hH1aMTSET
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Data Analysis
Similar to previous reports (Kellenberger et al., 1996;
Chahine et al., 1997; Vedantham and Cannon, 1998),
mutation of the phenylalanine to cysteine in the IFM
motif in the linker between domains III and IV caused
decay of INa to be moderately slowed compared with
wild-type hH1a, but perfusion with intracellular MTSET caused the decay to be almost completely inhibited (Fig. 2). We perfused cells expressing ICM-hH1a
internally with 2.5 mM MTSET until additional slowing
of the decay phase of INa in response to step depolarizations was detectable (typically within 4 min), at which
time the internal solution was changed back to control.
Over the next 6 min, further modification occurred before decay of INa stabilized with minimal inactivation
over 50 ms (Fig. 2 C). As reported for other mammalian sodium channels (Yang and Horn, 1995; Kellenberger et al., 1996; Chahine et al., 1997), although not
for squid giant axon (Khodakhah et al., 1998), internal
perfusion with MTSET had no effect on INa in wild-type
hH1a (n ⫽ 2, data not shown). In the following experiments, we compared INa and Ig measurements of ICMhH1aMTSET to wild-type hH1a.
Published May 1, 2000
Figure 2. Families of leak and
capacity-corrected INa during
step depolarizations to potentials
between ⫺120 and ⫹40 mV from
a holding potential of ⫺150 mV
for a cell expressing wild-type
hH1a (A), and a cell expressing
ICM-hH1a (B) in control (top)
and after exposure to 2.5 mM
intracellular MTSET (bottom).
Cells Y4.02 and X5.02.
Development of Ionic Current Inactivation of hH1a-ICM
Sodium Channels after Intracellular MTSET
A standard two-step development of inactivation protocol was used to assess the time course of fast inactivation of INa (Fig. 4). Fig. 4 A (insert) illustrates the voltage-clamp protocol, which included a short voltage
clamp-back step to ⫺120 for 2 ms, which allowed any
open sodium channels to close, but did not allow for
recovery of inactivated channels. Over the 44-ms conditioning time, wild-type hH1a almost completely inactivated, while ICM-hH1aMTSET demonstrated little inactivation. Data for development times up to 1 s were fitted
with the sum of two exponentials:
–t
---τf
–t
---τs
Fractional Availability = A f e + A s e + k,
(2)
where the parameters determined by the fit were: ␶f,
the faster time constant, Af, the amplitude contribution
of ␶f, ␶s, the slower time constant, As, the amplitude
contribution of ␶s, and k (1 ⫺ Af ⫺ As), the noninactivating fraction, which was set to 0 for hH1a. Table I
shows the values from fits to data from wild-type hH1a,
ICM-hH1aMTSET, and unmodified ICM-hH1a. For wildtype hH1a, the faster time constant was short (2.1 ⫾ 0.3
ms) and accounted for most of the inactivation (83 ⫾
2%). In contrast, inactivation was almost completely
abolished for ICM-hH1aMTSET, with only 18% ⫾ 2%
(n ⫽ 4) of the current inactivated by 44 ms, and the faster
time constant (6.9 ⫾ 3.0 ms) was more than three times
longer than that for hH1a. Similar results were found
when cells were perfused with 10 mM MTSETi; only
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Gating Current Immobilization
Figure 3. Family of gating currents (top) and their integrals
(bottom) for typical fused cells expressing hH1a (A) and ICMhH1aMTSET (B) for step depolarizations between ⫺110 and ⫹40
mV from a holding potential of ⫺150 mV. Data are shown capacity
and leak corrected, digitally filtered at 15 kHz, and with every fifth
point plotted. (Cells Y3.03 and Y4.20.) (C) Voltage dependence of
the dominant time constants (see text) obtained from fits to Ig relaxations for five cells expressing hH1a channels (䊉) and for four
cells expressing ICM-hH1aMTSET (䊊). (D) Q-V relationships for
hH1a (䊉) and for ICM-hH1aMTSET (䊊). Data for each cell were
normalized to their own Qmax. Solid lines represent the means of
the best fits to each cell by a Boltzmann distribution (Eq. 1). The
parameters from the best fits showed that, for hH1a (n ⫽ 5 cells),
the V1/2 was ⫺54 ⫾ 4 mV, the slope factor was ⫺12.0 ⫾ 2.5 mV,
and the mean Qmax was 5.3 ⫾ 2.8 pC; while, for ICM-hH1aMTSET
(n ⫽ 4 cells), the V1/2 was ⫺55 ⫾ 4 mV with a slope factor of
⫺15.5 ⫾ 2.4 mV, and the mean Qmax was 8.9 ⫾ 3.8 pC.
16% ⫾ 6% (n ⫽ 4) of the current inactivated by 44 ms
(data not shown). The residual “fast” inactivation in
ICM-hH1aMTSET was unlikely to reflect residual ICMhH1a unmodified by MTSETi because the fast time
constant of unmodified ICM-hH1a was much shorter
(1.4 ⫾ 0.3 ms). In addition, although a second time
constant was present for each channel isoform, the
slow time constant for ICM-hH1aMTSET would contribute little to the small reduction in INa at 44 ms because
it was nearly 80-fold longer than the fast time constant.
OFF-gating Current Studies
To investigate the contribution of the putative inactivation lid to gating charge immobilization (i.e., the slow
return of gating charge during repolarization), Ig was
recorded during repolarization steps to ⫺180 mV after
conditioning to 0 mV for up to 44 ms in cells express-
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(Fig. 3 D). These data indicate that an intact putative
inactivation lid did not directly contribute to gating
charge upon depolarization (ON charge).
Published May 1, 2000
TABLE I
Comparison of Parameters from Two Exponential Fits to the Development of I Na Inactivation (see Fig. 4)
Amplitude
of fast tau
Na channel
n
Fast tau
ms
fraction
hH1a
7
2.1 ⫾ 0.3
0.83 ⫾ 0.02
ICM-hH1aMTSET
4
6.9 ⫾ 3.0
0.12 ⫾ 0.03
ICM-hH1a
6
1.4 ⫾ 0.3
0.50 ⫾ 0.06
Constant
ms
fraction
fraction
21.7 ⫾ 10.9
0.17 ⫾ 0.02
0
406 ⫾ 158
0.43 ⫾ 0.14
0.46 ⫾ 0.14
59 ⫾ 22
0.24 ⫾ 0.03
0.26 ⫾ 0.04
between the two channels (Fig. 7). Data are graphed in
a manner similar to those presented by Armstrong and
Bezanilla (1977), who first showed that the time course
in the reduction of the fast component in OFF charge
was the same as the time course of development of INa
inactivation. Because the sum of the charge in the fastand slow-time constants equals unity, the time course of
appearance of a slow component in OFF charge is
equivalent to the time course of reduction in the contribution by the fast component. The fraction of OFF
charge accounted for by the fast component calculated
for each development duration compared with the total OFF charge measured for the same development
duration for wild-type hH1a and ICM-hH1aMTSET is
shown in Fig. 7. With the shortest conditioning duration of 0.3 ms, all of the OFF charge was accounted for
in the fast component, but, as the conditioning duration was extended, the fast component accounted for a
smaller fraction of the OFF charge.
For hH1a, the fraction of OFF charge contributed by
the fast-time constant decreased with a time constant of
2.8 ⫾ 2.3 ms, which was similar to the time course of the
development of INa inactivation at 0 mV (2.1 ms, Table
I). It reached a steady state value of 47 ⫾ 10%, where
the slow component accounted for the remainder
(53%) of the total OFF charge. Surprisingly, in the absence of a normally functioning inactivation gate (i.e.,
Figure 4. Two-pulse development of INa inactivation for
wild-type hH1a (A) and ICMhH1aMTSET (B). The inset illustrates the voltage-clamp protocol and an example of a capacity
and leak-corrected INa trace from
a cell expressing hH1a. Peak INa
after various conditioning times
to 0 mV were normalized to the
peak INa in the absence of a conditioning step for each cell. The
solid lines were calculated from
the means of the parameters of
the individual fits to Eq. 2. Fitted
parameters are given in Table I.
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Sheets et al.
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ing wild-type hH1a and ICM-hH1aMTSET. The potential
of ⫺180 mV was selected for recording of Ig during repolarization (OFF-Ig) because the slow component became fast enough such that all of the OFF charge could
be measured over 10–15 ms (i.e., the OFF charge measurement equaled the ON charge measurement), but it
was still long enough that the slow and fast time constants of OFF-Ig were different by about an order of
magnitude and could be clearly separated using two exponential fits. Examples of OFF-Ig and their integrals
are shown for hH1a and ICM-hH1aMTSET in Fig. 5. The
presence of a slow component is more apparent by inspection of the integrals of OFF-Ig (bottom).
OFF-Ig were fit by a sum of up to two exponentials,
and the time constants sorted by speed. Fig. 6 shows
the results of this analysis for both hH1a and ICMhH1aMTSET. For this and subsequent experiments, results from five cells expressing hH1a were compared
with those from four cells expressing ICM-hH1aMTSET.
The values for both ␶f and ␶s were very similar for the
two groups with a faster time constant of ⬍0.4 ms and a
slower time constant of ⵑ2 ms. Note that no slow time
constant was detectable until the duration of the conditioning step was at least 0.7 ms for either channel type.
Even though the time constants of relaxations of
OFF-Ig traces for hH1a and ICM-hH1aMTSET were similar, the contributions to OFF charge were very different
Amplitude
of slow tau
Slow tau
Published May 1, 2000
Figure 6. Time constants of OFF-gating current relaxations during repolarization to ⫺180 mV after conditioning to 0 mV for 0.3–
44 ms. The holding potential was ⫺150 mV. Fast (A) and slow (B)
time constants obtained from fits to Eq. 2 for cells expressing wildtype hH1a (䊉) and ICM-hH1aMTSET (䊊). Time constants were similar except those at times longer than 10 ms, which tended to increase slightly in ICM-hH1aMTSET.
in ICM-hH1aMTSET), there still remained two time constants in OFF-Ig. The time course of the decrease in the
fraction of OFF charge accounted by the fast-time constant had a similar value (2.5 ⫾ 1.4 ms) to that for hH1a,
although the fraction of OFF charge that was accounted
for by the fast-time constant after longer conditioning
durations was 69 ⫾ 9%, while the fraction of OFF
charge accounted for by the slow component decreased
to 31% (P ⬍ 0.01 compared with wild-type hH1a by nonpaired t test). Analysis of the time course of the appearance of the slow component in the OFF charge gave
nearly equivalent values; time constants and amplitudes
were 2.1 ⫾ 1.9 ms and 52 ⫾ 10% (wild-type hH1a) and
3.6 ⫾ 3.0 ms and 33 ⫾ 3% (ICM-hH1aMTSET). In one
cell, we confirmed that wild-type hH1a perfused with intracellular MTSET showed no changes in OFF-Ig.
Figure 7. Fraction of OFF
charge associated with the fasttime constant for hH1a (A) and
ICM-hH1aMTSET (B), normalized
to the total OFF charge measured during the same repolarization step. Voltage-clamp protocols and cells were the same as
in Fig. 6. The solid lines were calculated from the means of the
parameters from single exponential fits to the data for each cell
according to Eq. 3.
–t
-----τQ
Qf
----------- = ( 1 – A f )e + A f ,
Q OFF
(3)
where fractional charge was calculated as the ratio of charge associated with the faster time constant (Qf) to the total OFF charge
at each time, and the parameters determined by the fit were: ␶Q, the time constant of the reduction in the contribution to OFF charge by
the fast time constant, and Af, the steady state amplitude of OFF charge associated with the fast time constant while 1 ⫺ Af represents the
steady state amplitude of the OFF charge associated with the slow-time constant. The parameters from the best fits for hH1a (n ⫽ 5 cells)
showed that ␶Q was 2.8 ⫾ 2.3 ms; Af, the fraction of OFF charge in the fast tau, was 0.47 ⫾ 0.10; and 1 ⫺ Af, the fraction of OFF charge in the
slow tau, was 0.53 ⫾ 0.10.* For ICM-hH1aMTSET (n ⫽ 4 cells), ␶Q was 2.5 ⫾ 1.4 ms, Af was 0.69 ⫾ 0.09, and 1 ⫺ Af was 0.30 ⫾ 0.09.* *P ⬍ 0.01.
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Figure 5. OFF-gating currents (top) and their integrals (bottom)
recorded during repolarization to ⫺180 mV after conditioning
steps to 0 mV for 0.7, 3, 7, and 15 ms for hH1a (A) and ICMhH1aMTSET (B). Also shown for each cell is the ON-Ig in response to
a step depolarization to 0 mV from a holding potential of ⫺150 mV.
Data are shown capacity and leak corrected, digitally filtered at 15
kHz, and with every fourth point plotted. (Cells Y3.03 and Y4.40.)
Published May 1, 2000
The Effect of Inhibition of Movement of the Domain IV–S4 on
Charge Immobilization
We have shown that Ap-A toxin slows inactivation of INa
by inhibiting movement of the voltage sensor formed
by the S4 of domain IV (Hanck and Sheets, 1995;
Sheets et al., 1999). We postulated, therefore, that this
toxin could be used to probe the contribution of the S4
in domain IV to the slow component of OFF charge in
the lid-modified mutant.
Because fast inactivation had already been modified
in ICM-hH1aMTSET cells, the application of Ap-A toxin
had little additional effect on INa decay (data not
shown). However, because Ap-A toxin also reduces
Qmax by about one-third (Sheets and Hanck, 1995,
1999), the Q-V relationship could be used to verify that
channel modification by toxin had occurred. Fig. 8
shows that Ap-A toxin reduced Qmax by about one-third
in both wild-type hH1a and ICM-hH1aMTSET. Furthermore, the slope factors and half-points of the Q-V relationships for the two channels were comparable. The
reduction in Qmax by Ap-A toxin in ICM-hH1aMTSET confirmed that the toxin still modified ICM-hH1aMTSET ON
charge similar to wild-type hH1a, even though inactivation of INa had already been slowed.
The role of the domain IV–S4 in charge immobilization was then investigated by recording OFF-Ig for both
hH1a and ICM-hH1aMTSET after modification by Ap-A
615
Sheets et al.
Figure 8. Effects of Ap-A toxin
on ON charge. Q-V relationships
for hH1a (䊉) and ICMhH1aMTSET (䊊) after modification by 1 ␮M Ap-A toxin. ON
charge was normalized to the
Qmax in control solution for each
cell. The dashed line and thick
line represent the fits to the Q-V
relationships for hH1a and ICMhH1aMTSET in control solutions,
respectively, from Fig. 3. The thin solid lines represent the mean of
the best fits to each cell by a Boltzmann distribution (Eq. 1). The parameters from the best fits showed that for hH1a (n ⫽ 5 cells), the
V1/2 was ⫺57 ⫾ 5 mV, the slope factor was ⫺14.3 ⫾ 2.9 mV, and the
mean normalized Qmax was 0.62 ⫾ 0.07 pC, while for ICM-hH1aMTSET
(n ⫽ 4 cells), the V1/2 was ⫺59 ⫾ 4 mV, with a slope factor of
⫺14.3 ⫾ 3.0 mV, and the mean normalized Qmax was 0.67 ⫾ 0.09 pC.
toxin. The same voltage protocol was used as previously; cells were depolarized to 0 mV for durations up
to 44 ms before repolarization to ⫺180 mV. OFF-Ig
were fit by a sum of up to two exponentials and sorted
by speed. In contrast to the findings in control solutions (Fig. 6), OFF-Ig for both hH1a and ICMhH1aMTSET were best fit by a single exponential at all
conditioning durations (Fig. 9). The time constants
clustered around 0.3 ms, and they were similar to the
fast-time constants recorded for both hH1a and ICMhH1aMTSET cells in control solutions (Fig. 6 A). A decrease in the amount of charge immobilization has
been previously reported in frog myelinated nerve after
exposure to site-3 toxins (Neumcke et al., 1985).
To compare the magnitude of OFF charge for the
two channels after toxin modification, the fraction of
OFF charge in Ap-A toxin was normalized to the maximal OFF charge (i.e., Qmax) measured for each cell in
the absence of toxin and plotted as a function of the
duration of the conditioning pulse at 0 mV. These data
are shown in Fig. 10 A for both wild-type hH1a and
ICM-hH1aMTSET. The time course of the increase in
OFF charge was nearly identical for both sodium channels with a time constant of ⵑ0.7 ms. Note that the largest fraction of OFF charge was almost 70%, consistent
with the reduction in gating charge by Ap-A toxin (Fig.
9). Both the absence of a slow component in the OFF
Figure 9. Time constants of
OFF-gating currents after modification by Ap-A toxin for cells expressing wild-type hH1a (䊉) and
ICM-hH1aMTSET (䊊). Voltage protocol and data analysis were the
same as in Fig. 6. In contrast to
the control condition, all OFF-Ig
were best fit by a single exponential. Note the similar values to the
very fast-time constant obtained
in the absence of toxin (Fig. 6 A).
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While it is possible that the slow-time constant in
ICM-hH1aMTSET was related to residual inactivation of
unmodified channels, this seems unlikely for the following reasons. First, the fast-time constant of INa inactivation in ICM-hH1aMTSET was more than threefold
longer than that in unmodified ICM-hH1a channels
(6.9 vs. 1.4 ms, see Table I). Second, if inactivation of
residual, non–MTSET-modified ICM-hH1a channels
were responsible for the fast-time constant that remained in the OFF charge, then the amount of OFF
charge accounting for the slow component should have
been much smaller than the measured 31%. After all
wild-type hH1a channels have been fast inactivated,
ⵑ47% of the OFF charge returned rapidly and 53% returned slowly. Applying a similar ratio to OFF charge of
ICM-hH1aMTSET, where ⬍20% of INa became inactivated
by 44 ms (Fig. 4), it would be expected that ⬍10% of
the charge should have become immobilized. Instead,
⬎30% of the charge was contained in the slow-time
constant. Lastly, the time constant of INa inactivation at
0 mV for ICM-hH1aMTSET (6.9 ms) did not correspond
to its time constant of development of immobilization
(2.5 ms). Consequently, it is more likely that one or
more of the four putative voltage sensors of ICMhH1aMTSET return to their repolarized conformation
with a slow-time constant even in the absence of an intact inactivation lid.
Published May 1, 2000
Figure 10. Fraction of OFF
charge normalized to the cell’s
maximal OFF charge in control
after a conditioning step to 0 mV
for durations up to 44 ms. Solid
lines were calculated using the
means of fitted parameters from
each cell to the following:
–t
----
τ
fractional Q = A  1 – e  , (4)


where the parameters of the fit
were the time constant (␶) of the
appearance of OFF charge and
A, the fractional amplitude returned. (A) Fractional Q was calculated as the ratio of OFF
charge at the times indicated in
Ap-A toxin to the maximal OFF
charge in control solutions for
hH1a (䊉) and ICM-hH1aMTSET (䊊). (B) Fractional Q was calculated as the ratio of OFF charge in the fast time constant at the times indicated in control solutions (see Fig. 7) to the maximal OFF charge in control for hH1a (䊉) and ICM-hH1aMTSET (䊊). Note that both the
amplitude and time course of OFF charge associated with the fast time constant for ICM-hH1a MTSET were comparable with those of both
toxin-modified channels. The parameters from the best fits are given in Table II.
D I S C U S S I O N
To study the role of putative inactivation lid on charge
immobilization, we investigated hH1a that had the phenylalanine at position 1485 in the IFM region of the domain III–IV linker mutated to a cysteine (F1485C). Although the mutation itself has been shown to only
moderately disrupt INa inactivation in three different
mammalian sodium channel isoforms (rat brain IIa,
skeletal muscle, and heart), fast inactivation is almost
completely eliminated by exposure of the mutated cysteine to intracellular MTSET (Kellenberger et al., 1996;
Chahine et al., 1997; Vedantham and Cannon, 1998).
We used this approach to study the contribution of binding of the inactivation lid to gating charge immobilization in fused mammalian cells expressing ICM-hH1a. Al-
TABLE II
Parameters from Fits to Eq. 4 for Fraction of OFF Charge Normalized to Maximal OFF Charge in Control
Na channel isoform
n
Toxin present
A
(fractional
amplitude)
hH1a
5
yes
0.69 ⫾ 0.08
0.74 ⫾ 0.15
ICM-hH1aMTSET
4
yes
0.69 ⫾ 0.06
0.98 ⫾ 0.17
Fast component in ICM-hH1aMTSET
4
no
0.71 ⫾ 0.09
0.74 ⫾ 0.24
Fast component in hH1a
5
no
0.54 ⫾ 0.07
0.42 ⫾ 0.10
Tau
ms
There was no significant difference (P ⬍ 0.05) between the amplitudes and taus of OFF charge for ICM-hH1aMTSET in toxin and either wild-type hH1a in
toxin or OFF charge associated with the fast time constant of ICM-hH1aMTSET in control solution.
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Gating Current Immobilization
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counted for by the fast component for wild-type hH1a
recorded in control solutions.
charge of ICM-hH1aMTSET after toxin modification and
the ⵑ30% reduction in OFF charge suggest that the
slow component of OFF charge for ICM-hH1aMTSET in
control solutions (Fig. 7) may result from the slow return of charge associated with the S4 in domain IV.
If this were the case, then one would expect the OFF
charge associated with the fast-time constant in ICMhH1aMTSET to be similar to the OFF charge of both
toxin-modified ICM-hH1aMTSET and toxin-modified
wild-type hH1a. Fig. 10 B shows the time course of the
appearance of OFF charge contributed by the fast time
constant in control solutions for both wild-type hH1a
and ICM-hH1aMTSET normalized to maximal OFF
charge (i.e., Qmax). Note that the fraction and time
course of OFF charge contributed by the fast-time constant in ICM-hH1aMTSET recorded in control solutions
was nearly identical to that for toxin-modified ICMhH1aMTSET. Also shown in Fig. 10 B is the time course
and magnitude of the fraction of OFF charge ac-
Published May 1, 2000
though fast inactivation had been modified in this preparation, inactivation over hundreds of milliseconds can
still occur in sodium channels with mutations in the IFM
motif (Featherstone et al., 1996). Such slow inactivation
was not addressed in the studies reported here.
We found that the Q-V relationships of wild-type
hH1a and ICM-hH1aMTSET were similar, as were the
time courses of ON-Ig relaxations, demonstrating that
an intact inactivation lid had little effect on movement
of the channel’s voltage sensors during step depolarizations. However, comparison of the OFF-charge measurements between the two sodium channels showed
that 53% of the charge became immobilized in wildtype hH1a, while only 31% of the charge became immobilized in ICM-hH1aMTSET. After the application of
Ap-A toxin, a site-3 peptide toxin that has been shown
to modify inactivation of INa by inhibiting movement of
the S4 segment of domain IV (Sheets et al., 1999), the
OFF charge was similar in both magnitude and time
course for both wild-type hH1a and ICM-hH1aMTSET.
Armstrong and Bezanilla (1977) found that the OFF
charge of inactivated sodium channels in squid giant
axon contained two components, a fast and a slow component, with the slow component (immobilizable fraction) accounting for nearly 60% of the total charge.
Furthermore, the time course of the reduction in the
fast component in OFF charge (as well as the time
course of the development of the slow component)
paralleled the time course of development of INa inactivation. When the repolarization voltage was less negative, the slow component of the OFF charge was too
small and too slow to be accurately measured. As a consequence, the magnitude of the OFF charge was reduced compared with ON charge, and the gating
charge was said to become immobilized. We found a
similar correlation between the time course of reduction in the fast component of OFF charge and the degree of inactivation of INa. In wild-type hH1a, the predominant time constant of INa inactivation at 0 mV was
2.1 ms, which was comparable with both the time
course of reduction in the fast component (2.8 ms) and
to the appearance of the slow component (2.1 ms) in
OFF charge (Figs. 4 and 7). After longer conditioning
durations, the slow component accounted for up to
53% of total OFF charge, similar to the nearly 60%
found for sodium channels in squid giant axon (Armstrong and Bezanilla, 1977; Greeff et al., 1982) and to
the 56% found for heterologously expressed rat brain
IIa (Kuhn and Greeff, 1999).
Armstrong and Bezanilla (1977) also found that internal perfusion of the squid giant axon with the nonspecific proteolytic enzyme, pronase, removed both fast
inactivation and charge immobilization. Based on this
617
Sheets et al.
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The Role of the Inactivation Lid in Charge Immobilization
finding, it seemed reasonable to expect that focal disruption of the putative inactivation lid of sodium channels might also eliminate charge immobilization. However, we found that was not the case; the amount of
OFF charge in ICM-hH1aMTSET that could become immobilized was reduced but not eliminated (from 53%
to 31%). In ICM-hH1aMTSET, charge immobilization still
occurred with a time course of 2.5 ms (Fig. 7), which
was similar to the time course in wild-type hH1a. Although fast inactivation was “removed” by either intracellular proteolytic enzymes or by mutagenesis, these
results suggest that the two methods are not equivalent.
Similarly, differences between the two methods has
been shown for cardiac Na channels where modification of fast inactivation by proteolytic enzymes enhanced slow inactivation (Clarkson, 1990), whereas
mutation of the inactivation lid had little effect on slow
inactivation (Bennett et al., 1995). Because proteolytic
enzymes are expected to cleave the Na channel protein
at multiple sites, it is likely that intracellular proteolytic
enzymes produce more complicated channel modifications than that achieved by channel mutagenesis. Consequently, differing effects on movement of the voltage
sensors would not be unexpected.
In the absence of binding of the inactivation lid, what
then is the origin of the gating charge that can become
immobilized in ICM-hH1aMTSET? Fig. 10 suggests that
the S4 in domain IV is responsible for charge immobilization in ICM-hH1aMTSET. Previous studies have shown
that site-3 toxins such as Ap-A toxin bind extracellularly
to regions in domain IV (Thomsen and Catterall, 1989;
Rogers et al., 1996; Benzinger et al., 1997, 1998), and
that site-3 toxins inhibit movement of the S4 of domain
IV (Sheets and Hanck, 1995; Sheets et al., 1999). Consequently, sodium channels that are modified by Ap-A
toxin allow only the S4s in domains I–III to contribute
to gating charge. In Ap-A toxin, the time course and
the magnitude of OFF charge for both wild-type hH1a
and ICM-hH1aMTSET were the same (Fig. 10 A). Comparison of these data to the OFF charge associated with
only the fast-time constant in ICM-hH1aMTSET in control
solutions showed the time courses and magnitudes of
the OFF charge to be analogous (Fig. 10 B), and suggests that the S4s in domains I–III in nontoxin-modified ICM-hH1aMTSET contributed to the fast component
of OFF charge. Consequently, the S4 in domain IV appears to be responsible for the slow component of OFF
charge in ICM-hH1aMTSET.
These data also suggest that charge movement from
the S4 in domain IV occurs on a slower time scale than
activation. In the presence of Ap-A toxin, all OFF charge
is contained in the fast component with a time course
of appearance of ⵑ0.7 ms (Fig. 10), and represents the
return of gating charge associated only with channel
activation because fast inactivation has been inhibited
Published May 1, 2000
Implications for a Structurally Based Model
The experiments reported here expand upon the
model recently proposed by Cha et al. (1999). Our
studies confirm that the S4 in domain IV is the last voltage sensor to move during repolarization after fast inactivation, and that it contributes ⵑ30% to the total OFF
charge. The results in this study demonstrate that the
slow movement of the S4 in domain IV during repolarization is intrinsic to the voltage sensor, and not dependent on the binding of the inactivation lid. In the
model proposed by Cha el al. (1999), the rate-limiting
step during repolarization was proposed to be the unbinding of the inactivation lid. If that were the case,
then one would expect either no slow component in
the OFF charge of ICM-hH1aMTSET or a component that
was intermediate between the fast and slow components of wild-type hH1a. But this was not what was
found; the time course of the slow component in the
OFF charge in ICM-hH1aMTSET had the same time course
618
Gating Current Immobilization
as that of INa inactivation. This finding suggests that the
rate-limiting step is not the unbinding of the inactivation lid, but the movement of the S4 in domain IV.
However, binding of the inactivation lid did modulate
the movement of an additional voltage sensor that contributed 22% to the OFF charge; i.e., the difference between the 53% of charge immobilized in wild-type
hH1a and the 31% of charge immobilized in ICMhH1aMTSET that could be attributed to the S4 in domain
IV. Our data taken together with the data of Cha et al.
(1999) suggest that a likely candidate for this is the S4
in domain III. When the inactivation lid is not bound to
its receptor, either because the inactivation lid has been
altered, as in ICM-hH1aMTSET, or because the receptor
for the inactivation lid has been modified by inhibition
of the movement of the domain IV–S4 by site-3 toxins
in wild-type hH1a, the S4 in domain III moves rapidly
and contributes to the fast component of OFF charge.
We thank WenQing Yu for her excellent technical assistance.
This research was supported by National Heart, Lung and
Blood Institute grants HL-PO1-20592 (D.A. Hanck and J.W. Kyle)
and HL-R01-44630 (M.F. Sheets).
Submitted: 29 November 1999
Revised: 7 March 2000
Accepted: 10 March 2000
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Published May 1, 2000
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